Methods of assessing HIV integrase inhibitor therapy

ABSTRACT

Methods and products for the evaluation of HIV treatment are provided. The methods are based on evaluating molecular events at the HIV integrase resulting in altered therapeutic efficacy of tho investigated compounds. The methods rely on providing an integrase gene and evaluating either through integrase gene genotyping or phenotyping.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application claims priority to U.S. Provisional Application Ser.No. 60/310,480, filed Aug. 8, 2001, and EP Application No. 01203012.8,filed Aug. 8, 2001, all of which are incorporated herein by reference intheir entirety.

The present invention relates to methods and products for evaluatingtreatment of human immunodeficiency virus (HIV). In particular,molecular events at HIV integrase and their effect on therapeuticefficacy of drugs are determined. Suitably, the events are analysed bygenotyping or phenotyping of HIV integrase. The methods and productsdescribed herein find use in multiple fields including diagnostics, drugscreening, pharmacogenetics and drug development.

Several different treatment regimens have been developed to combat HIVinfection. However, since the HIV virus is mutating quickly, becausereverse transcriptase (RT) duplicating the genetic material has noproofreading capacity, it can counter the effects of drugs or drugcombinations used against it. Current HIV chemotherapy involvesinhibitors of the reverse transcriptase (RT) and protease enzymes.Despite the development of novel classes of inhibitors and complex drugregimens, drug resistance is increasing. Thus, new types of anti-HIVdrugs are continually necessary. Development of compounds that inhibitother HIV gene products in vivo such as the envelope, tat, and integrase(IN) is a key area of investigation.

The integrase protein represents a target for HIV inhibitor research.HIV integrase is required for integration of the viral genome into thegenome of the host cell, a step in the replicative cycle of the virus.It is a protein of about 32 KDa encoded by the pol gene, and is producedin vivo by protease cleavage of the gag-pol precursor protein during theproduction of viral particles. The integration process takes placefollowing reverse transcription of the viral RNA. First, the viralintegrase binds to the viral DNA and removes two nucleotides from the 3′end of the viral long-terminal repeat (LTR) sequences on each strand.This step is called 3′ end processing and occurs in the cytoplasm withina nucleoprotein complex termed the pre-integration complex (PIC).Second, in a process called strand transfer, the two strands of thecellular DNA into which the viral DNA will be inserted, i.e. the targetDNA, are cleaved in a staggered fashion. The 3′ ends of the viral DNAare ligated to the 5′ ends of the cleaved target DNA. Finally, remaininggaps are repaired, probably by host enzymes.

With the increasing number of available anti-HIV compounds, the numberof potential treatment protocols for HIV infected patients will continueto increase. Many of the currently available compounds are administeredas part of a combination therapy. The high complexity of treatmentoptions coupled with the ability of the virus to develop resistance toHIV inhibitors requires the frequent assessment of treatment strategies.The ability to accurately monitor the replicative capacity of viruses inpatients subjected to a drug regimen and to use that data to modify thedoses or combinations of inhibitors allows physicians to effectivelyreduce the formation of drug resistant virus and provide an optimal,tailored treatment for each patient.

Sophisticated patient monitoring techniques have been developed foranalysis of current therapies, e.g. such as Antivirogram®, (described inWO 97/27480 and U.S. Pat. No. 6,221,578 B1; incorporated herein byreference) and Phenosense™ (WO 97/27319). These cellular based assaysdetermine the resistance of the patient borne virus towards a defineddrug regimen by providing information about the susceptibility of thepatient's virus strain to the treatment based on protease and reversetranscriptase inhibitors treatment. Other monitoring strategies includeimmunological means or sequencing techniques.

The Antivirogram® and Genseq™ assays determine the phenotype andgenotype respectively of a patient's reverse transcriptase and proteasegenes. The relevant coding regions are obtained from patient samples,reverse transcribed and amplified by the polymerase chain reaction(PCR). Within lymphocyte cells the relevant coding regions are combinedwith viral deletion constructs to create chimeric viruses. The abilityof these chimeric viruses to invade and kill cells in culture isassessed in the presence of HIV reverse transcriptase and proteaseinhibitors. A database combining phenotypic and genotypic informationcan be developed, as described in WO 00/73511 (incorporated herein byreference).

While phenotyping and genotyping assays such as Antivirogram® andGenseq™ have been developed for reverse transcriptase and proteasegenes, protocols for evaluation of drug resistance at the integrase genehave not been successfully developed.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides techniques for evaluating humanimmunodeficiency (HIV) drug effectiveness. Assays for wild type ormutant HIV integrase are provided, using a set of primers designed forthe amplification and analysis of HIV genetic material. The assessmentof patient borne viral integrase leads to a better prediction of thedrugs suitable for treatment of the strains present in the infectedindividual. The protocols and products may be used for diversediagnostic, clinical, toxicological, research and forensic purposesincluding, drug discovery, designing patient therapy, drug efficacytesting and patient management. The assays described herein may be usedin combination with other assays. The results may be implemented incomputer models and databases. The products described herein may beincorporated into kits.

The instant invention relates to a method for determining thesusceptibility of at least one HIV virus to at least one treatment,comprising: i) obtaining at least one sample of HIV RNA, wherein thesample comprises at least one IN gene or a portion thereof; ii)reverse-transcribing and amplifying the HIV RNA with primers specificfor IN region of the HIV genome to obtain at least one DNA constructcomprising the at least one IN gene or a portion thereof; iii) preparingat least one recombinant virus by homologous recombination or ligationbetween the amplified at least one DNA construct and a plasmidcomprising the wild-type HIV sequence with a deletion in the IN regionof the HIV genome, and iv) determining the phenotypic susceptibility ofat least one HIV virus to at least one treatment by monitoring the atleast one recombinant virus in the presence of the at least onetreatment.

In particular, the present invention relates to a method for determiningthe susceptibility of at least one HIV virus to at least one drug,comprising: i) obtaining at least one sample comprising HIV RNA, whereinthe sample comprises at least one IN gene or a portion thereof; ii)reverse transcribing and amplifying the HIV RNA with primers specificfor IN region of the HIV genome to obtain at least one ampliconcomprising the at least one IN gene or a portion thereof; iii) usingnucleic acid amplification to generate a plasmid comprising thewild-type HIV sequence with a deletion in the IN region of the HIVgenome; iv) preparing at least one recombinant virus by homologousrecombination or ligation between the amplified at least one ampliconand a plasmid comprising the wild-type HIV sequence with a deletion inthe IN region, and v) monitoring the at least one recombinant virus inthe presence of the at least one treatment to determine the phenotypicsusceptibility of at least one HIV virus to said at least one drug.

Reverse transcription and amplification may be performed with a singleset of primers. Alternatively, more than one set of primers may be usedin a hemi-nested approach to reverse transcribe and amplify the geneticmaterial. Particularly, more than one set of primer is used in a nestedapproach. Following the generation of the recombinant construct, thechimeric virus may be grown and the viral titer determined (expressed asmultiplicity of infection, MOI) before proceeding to the determinationof the phenotypic susceptibility. The indicator gene, encoding a signalindicative of replication of the virus in the presence of a drug orindicative of the susceptibility of the virus in the presence of a drugmay be present in the culturing cells such as MT-4 cells. In addition,said indicator gene may be incorporated in the chimeric constructintroduced into the culturing cells or may be introduced separately.Suitable indicator genes encode fluorescent proteins, particularly greenfluorescent protein or mutants thereof. In order to allow homologousrecombination, genetic material may be introduced into the cells using avariety of techniques known in the art including, calcium phosphateprecipitation, liposomes, viral infection, and electroporation. Themonitoring may be performed in high throughput.

A human immunodeficiency virus (HIV), as used herein refers to any HIVincluding laboratory HIV strains, wild type HIV strains, mutant HIVstrains and any biological sample comprising at least one HIV virus,such as, for example, an HIV clinical isolate. HIV strains compatiblewith the present invention are any such strains that are capable ofinfecting mammals, particularly humans. Examples are HIV-1 and HIV-2.For reduction to practice of the present invention, an HIV virus refersto any sample comprising at least one HIV virus. As for instance apatient may have HIV viruses in his body with different mutations in theintegrase (IN) gene. It is to be understood that a sample may contain avariety of different HIV viruses containing different mutationalprofiles in the IN gene. A sample may be obtained for example from anindividual, from cell cultures, or generated using recombinanttechnology, or cloning. HIV strains compatible with the presentinvention are any such strains that are capable of infecting mammals,particularly humans. Viral strains used for obtaining a plasmid arepreferably HIV wild-type sequences, such as LAI or HXB2D. LAI, alsoknown as IIIB, is a wild type HIV strain. One particular clone thereof,this means one sequence, is HXB2D. This sequence may be incorporatedinto a plasmid.

Instead of viral RNA, HIV DNA, e.g. proviral DNA, may be used for themethods described herein. In case RNA is used, reverse transcriptioninto DNA by a suitable reverse transcriptase is needed. The protocolsdescribing the analysis of RNA are also amenable for DNA analysis.However, if a protocol starts from DNA, the person skilled in the artwill know that no reverse transcription is needed. The primers designedto amplify the RNA strand, also anneal to, and amplify DNA. Reversetranscription and amplification may be performed with a single set ofprimers. Suitably a hemi-nested and more suitably a nested approach maybe used to reverse transcribe and amplify the genetic material.

Thus, the phenotyping method of the present invention may also comprise:i) obtaining at least one sample comprising HIV DNA, wherein the samplecomprises at least one IN gene or a portion thereof; ii) amplifying theHIV DNA with primers specific for IN region of the HIV genome to obtainat least one amplicon comprising the at least one IN gene or a portionthereof; iii) generating a plasmid comprising the wild-type HIV sequencewith a deletion in the IN region of the HIV genome characterized in thatsaid deletion is generated using nucleic acid amplification; iv)preparing at least one recombinant virus by homologous recombination orligation between the amplified at least one amplicon and a plasmidcomprising the wild-type HIV sequence with a deletion in the IN region,and v) monitoring the at least one recombinant virus in the presence ofthe at least one drug to determine the phenotypic susceptibility of atleast one HIV virus to at least one drug.

Nucleic acid may be amplified by techniques such as polymerase chainreaction (PCR), nucleic acid sequence based amplification (NASBA),self-sustained sequence replication (3SR), transcription basedamplification (TAS), ligation chain reaction (LCR). Often PCR is used.

Any type of patient sample may be used to obtain the integrase gene,such as, for example, serum and tissue. Viral RNA may be isolated usingknown methods such as described in Boom, R. et al. (J. Clin. Microbiol.28(3): 495-503 (1990)). Alternatively, a number of commercial methodssuch as the QIAAMP® viral RNA kit (Qiagen, Inc.) may be used to obtainviral RNA from bodily fluids such as plasma, serum, or cell-free fluids.DNA may be obtained by procedures known in the art (e.g. Maniatis, 1989)and commercial procedures (e.g. Qiagen).

The complete integrase (IN) or a portion of the IN gene may be used. Thecomplete IN gene comprises 864 nucleotides (nt), coding for a 288 aminoacid long integrase. A portion of the IN gene is defined as a fragmentof IN gene recovered from patient borne virus, lab viruses includingIIIB and NL4-3, or mutant viruses. This fragment does not encompass thecomplete 864 nt. Said fragment may be obtained directly from its source,including a patient sample, or may be obtained using molecular biologytools following the recovery of the complete IN sequence. Ampliconrefers to the amplified, and where necessary, reverse transcribedintegrase gene or portion thereof. It should be understood that this INmay be of diverse origin including plasmids and patient material.Suitably, the amplicon is obtained from patient material. For thepurpose of the present invention the amplicon is sometimes referred toas “DNA construct”. A viral sequence may contain one or multiplemutations versus the consensus reference sequence given by K03455. Saidsequence, K03455, is present in Genbank and available through theinternet. A single mutation or a combination of IN mutations maycorrelate to a change in drug efficacy. This correlation may beindicative of an altered i.e. decreased or increased susceptibility ofthe virus for a drug. Said mutations may also influence the viralfitness.

“Chimeric” means a construct comprising nucleic acid material fromdifferent origin such as for example a combination of wild type HIV witha laboratory HIV virus, a combination of wild type HIV sequence andpatient derived HIV sequence.

A “drug” means any agent such as a chemotherapeutic, peptide, antibody,antisense, ribozyme and any combination thereof. Examples of drugsinclude protease inhibitors including ritonavir, amprenavir, nelfinavir;reverse transcriptase inhibitors such as nevirapine, delavirdine, AZT,zidovudine, didanosine; integrase inhibitors; agents interfering withenvelope (such as for example T-20, T-1249). Treatment or treatmentregimen refers to the therapeutic management of an individual by theadministration of drugs. Different drug dosages, administration schemes,administration routes and combinations may be used to treat anindividual.

An alteration in viral drug sensitivity is defined as a change insusceptibility of a viral strain to said drug. Susceptibilities aregenerally expressed as ratios of EC₅₀ or EC₉₀ values (the EC₅₀ or EC₉₀value being the drug concentration at which 50% or 90% respectively ofthe viral population is inhibited from replicating) of a viral strainunder investigation compared to the wild type strain. Hence, thesusceptibility of a viral strain towards a certain drug can be expressedas a fold change in susceptibility, wherein the fold change is derivedfrom the ratio of for instance the EC₅₀ values of a mutant viral straincompared to the wild type EC₅₀ values. In particular, the susceptibilityof a viral strain or population may also be expressed as resistance of aviral strain, wherein the result is indicated as a fold increase in EC₅₀as compared to wild type EC₅₀. The IC₅₀ is the drug concentration atwhich 50% of the enzyme activity is inhibited.

The susceptibility of at least one HIV virus to a drug may be tested bydetermining the cytopathogenicity of the recombinant virus to cells. Inthe context of this invention, the cytopathogenic effect means theviability of the cells in culture in the presence of chimeric viruses.The cells may be chosen from T cells, monocytes, macrophages, dendriticcells, Langerhans cells, hematopoetic stem cells or precursor cells, MT4cells and PM-1 cells. Suitable host cells for homologous recombinationof HIV sequences include MT4 and PM-1. MT4 is a CD4⁺ T-cell linecontaining the CXCR4 co-receptor. The PM-1 cell line expresses both theCXCR4 and CCR5 co-receptors. All of the cells mentioned above arecapable of producing new infectious virus particles upon recombinationof the IN deletion vectors with IN sequences such as those derived frompatient samples. Thus, they can also be used for testing thecytopathogenic effects of recombinant viruses. The cytopathogenicitymay, for example, be monitored by the presence of any reporter moleculeincluding reporter genes. A reporter gene is defined as a gene whoseproduct has reporting capabilities. Suitable reporter molecules includetetrazolium salts, green fluorescent proteins, beta-galactosidase,chloramfenicol transferase, alkaline phophatase, and luciferase. Severalmethods of cytopathogenic testing including phenotypic testing aredescribed in the literature comprising the recombinant virus assay(Kellam and Larder, Antimicrob. Agents Chemotherap. 1994, 38, 23-30,Hertogs et al. Antimicrob. Agents Chemotherap. 1998, 42, 269-276;Pauwels et al. J. Virol Methods 1988, 20, 309-321)

The susceptibility of at least one HIV virus to at least one drug may bedetermined by the replicative capacity of the recombinant virus in thepresence of at least one drug, relative to the replicative capacity ofan HIV virus with a wild-type IN gene sequence. Replicative capacitymeans the ability of the virus or chimeric construct to grow underculturing conditions. This is sometimes referred to as viral fitness.The culturing conditions may contain triggers that influence the growthof the virus, examples of which are drugs. The methods for determiningthe susceptibility may be useful for designing a treatment regimen foran HIV-infected patient. For example, a method may comprise determiningthe replicative capacity of a clinical isolate of a patient and usingsaid replicative capacity to determine an appropriate drug regime forthe patient. One approach is the Antivirogram® assay.

The IN phenotyping assays of the present invention can be performed athigh throughput using, for example, a microtiter plate containing avariety of anti-HIV drugs. The present assays may be used to analyse theinfluence of changes at the HIV IN gene to any type of drug useful totreat HIV. Examples of anti-HIV drugs that can be tested in this assayinclude, nucleoside and non-nucleoside reverse transcriptase inhibitors,nucleotide reverse transcriptase inhibitors, protease inhibitors,membrane fusion inhibitors, and integrase inhibitors, but those of skillin the art will appreciate that other types of antiviral compounds mayalso be tested. The results may be is monitored by several approachesincluding but not limited to morphology screening, microscopy, andoptical methods, such as, for example, absorbance and fluorescence. AnIC₅₀ value for each drug may be obtained in these assays and used todetermine viral replicative capacity in the presence of the drug. Apartfrom IC₅₀ also e.g. IC₉₀ or EC₅₀ (effective concentrations) can be used.The replicative capacity of the viruses may be compared to that of awild-type HIV virus to determine a relative replicative capacity value.Data from phenotypic assays may further be used to predict the behaviourof a particular HIV isolate to a given drug based on its genotype.

The assays of the present invention may be used for therapeutic drugmonitoring. Said approach includes a combination of susceptibilitytesting, determination of drug level and assessment of a threshold. Saidthreshold may be derived from population based pharmacokinetic modelling(WO 02/23186). The threshold is a drug concentration needed to obtain abeneficial therapeutic effect in vivo. The in vivo drug level may bedetermined using techniques such as high performance liquidchromatography, liquid chromatography, mass spectroscopy or combinationsthereof. The susceptibility of the virus may be derived from phenotypingor interpretation of genotyping results i.e. virtual phenotyping (WO01/79540).

The assays of the present invention may be useful to discriminate aneffective drug from an ineffective drug by establishing cut-offs i.e.biological cut-offs (see e.g. WO 02/33402). A biological cut-off is drugspecific. These cut-offs are determined following phenotyping a largepopulation of individuals containing wild type viruses. The cut-off isderived from the distribution of the fold increase in resistance of thevirus for a particular drug.

The instant invention also relates to a kit for phenotyping HIVintegrase. Such kit, useful for determining the susceptibility of atleast one HIV virus to at least one drug, may comprise: i) at least oneof the primers selected from SEQ ID N^(o)1-16, and ii) a plasmid asdescribed in the present invention. For the purpose of performing thephenotyping assay, such kit may be further completed with at least oneinhibitor. Optionally, a reference plasmid bearing a wild type HIVsequence may be added. Optionally, cells susceptible of HIV transfectionmay be added to the kit. In addition, at least one reagent formonitoring the indicator genes, or reporter molecules such as enzymesubstrates, may be added.

The present invention also describes a method for determining thesusceptibility of at least one HIV virus to at least one drug,comprising: i) obtaining at least one sample comprising HIV RNA, whereinthe sample comprises at least one IN gene or a portion thereof; ii)reverse-transcribing and amplifying said HIV RNA with primers specificfor the IN region of the HIV genome to obtain an amplicon comprising theIN gene or a portion thereof; iii) determining the nucleotide sequenceof the amplicon or a portion thereof, and iv) comparing the nucleotidesequence of the amplicon to the sequence of known sequences to determinethe susceptibility of at least one HIV virus to at least one drug. Thisassay protocol is commonly referred to as genotyping.

The genotype of the patient-derived IN coding region may be determineddirectly from the amplified DNA, i.e. the DNA construct, by performingDNA sequencing during the amplification step. Alternatively, thesequence may be obtained after sub-cloning into a suitable vector. Avariety of commercial sequencing enzymes and equipment may be used inthis process. The efficiency may be increased by determining thesequence of the IN coding region in several parallel reactions, eachwith a different set of primers. Such a process could be performed athigh throughput on a multiple-well plate, for example. Commerciallyavailable detection and analysis systems may be used to determine andstore the sequence information for later analysis. The nucleotidesequence may be obtained using several approaches including sequencingnucleic acids. This sequencing may be performed using techniquesincluding gel based approaches, mass spectroscopy and hybridisation.However, as more resistance related mutations are identified, thesequence at particular nucleic acids, codons or short sequences may beobtained. If a particular resistance associated mutation is known, thenucleotide sequence may be determined using hybridisation assays(including Biochips, LipA-assay), mass spectroscopy, allele specificPCR, or using probes or primers discriminating between mutant andwild-type sequence. For these purposes the probes or primers may besuitably labelled for detection (e.g. Molecular beacons, TaqMan®,SunRise primers). Suitably, fluorescent or quenched fluorescent primersare used. The primer is present in a concentration ranging from 0.01pmol to 100 pmol, suitably between 0.10 and 10 pmol. The cyclingconditions include a denaturation step during 0.5 to 10 minutes,suitably, 1 to 5 minutes at a temperature ranging from 85 to 99° C.Interestingly, the temperature is between 90 and 98° C. Subsequently,the material is cycled during 14 to 45 cycles, suitably between 20 to 40cycles, more suitably during 25 to 35 cycles. Nucleic acid is denaturedat 90 to 98° C. during 5 seconds to 2 minutes. Suitably, denaturationperiods range from 15 seconds to 1 minute. Annealing is performed at 40to 60° C., specifically, between 45° C. and 57° C. The annealing periodis 5 seconds to 1 minute, especially between 10 seconds and 35 seconds.Elongation is performed at 60° C. to 75° C. during 10 seconds to 10minutes. Preferably, the elongation period is 15 seconds to 5 minutes. Aselected set of sequencing primers includes SEQ ID 17-22. Thisparticular selection has the advantage that it enables the sequencing ofthe complete HIV integrase gene. Consequently, using this set of primersall possible mutations that may occur in the HIV integrase gene may beresolved.

The patient IN genotype provides an additional means to determine drugsusceptibility of a virus strain. Phenotyping is a lengthy process oftenrequiring 2 or more weeks to accomplish. Therefore, systems have beendeveloped which enable the prediction of the phenotype based on thegenotypic results. The results of genotyping may be interpreted inconjunction with phenotyping and eventually be subjected to databaseinterrogation. A suitable system is virtual phenotyping (WO 01/79540).In the virtual phenotyping process the complete IN genes may be used.Alternatively, portions of the genes may be used. Also combinations ofmutations, preferentially mutations indicative of a change in drugsusceptibility, may be used. A combination of mutations is sometimesreferred to as a hot-spot (see e.g. WO 01/79540). Briefly, in theprocess of virtual phenotyping, the genotype of a patient derived INsequence may be correlated to the phenotypic response of said patientderived IN sequence. If no phenotyping is performed, the sequence may bescreened towards a collection of sequences present in a database.Identical sequences are retrieved and the database is furtherinterrogated to identify if a corresponding phenotype is known for anyof the retrieved sequences. In this latter case a virtual phenotype maybe determined. A report may be prepared including the EC₅₀ of the viralstrain for one or more therapies, the sequence of the strain underinvestigation, biological cut-offs.

The present invention also relates to a kit for genotyping HIVintegrase. Such kit useful for determining the susceptibility of atleast one HIV virus to at least one drug may comprise at least oneprimer selected from SEQ ID N^(o) 1-12 and 17-22. Optionally, additionalreagents for performing the nucleic amplification and subsequentsequence analysis may be added. Reagents for cycle sequencing may beincluded. The primers may be fluorescently labelled.

The instant invention provides a method of identifying a drug effectiveagainst HIV integrase comprising: i) obtaining at least one HIVintegrase sequence, ii) determining the phenotypic response of theintegrase towards said drug, iii) using said phenotypic response todetermine the effectiveness of said drug. The phenotypic response isdetermined according to the methods of the instant invention.

The methods described in the instant invention may be used in a methodof identifying a drug effective against HIV integrase comprising: i)obtaining at least one HIV integrase sequence, determining the sequenceof said HIV integrase, ii) comparing said sequence with sequencespresent in a database of which the susceptibility has been determined ofthe HIV integrase, iii) using said sequence comparison to determine theeffectiveness of said drug. The susceptibility and the sequence of theHIV integrase gene are determined according to the methods disclosed inthe instant invention.

The genotyping and phenotyping methods as described herein can be usedto create a genotypic and phenotypic database of IN sequences,comprising: i) obtaining samples comprising HIV RNA comprising the INgene or a portion thereof; ii) reverse-transcribing and amplifying saidHIV RNA with primers specific for the IN region of the HIV genome toobtain an amplicon comprising the IN gene or a portion thereof; iii)determining the nucleotide sequence of the amplicon or portions thereof;iv) generating a plasmid comprising the wild-type HIV sequence with adeletion in the IN region of the HIV genome characterized in that saiddeletion is generated using nucleic acid amplification; v) preparingrecombinant virus by homologous recombination or ligation between theamplicon and a plasmid comprising the wild-type HIV sequence with adeletion in the IN region of the HIV genome, characterised in that saiddeletion is introduced using PCR; vi) determining the relativereplicative capacity of the recombinant virus in the presence ofanti-HIV drugs compared to an HIV virus with a wild-type IN genesequence; vii) correlating the nucleotide sequence and relativereplicative capacity in a data table.

According to the methods described herein a database may be constructedcomprising genotypic and phenotypic data of the HIV integrase, whereinthe database further provides a correlation between genotypes andbetween genotypes and phenotypes, wherein the correlation is indicativeof efficacy of a given drug regimen. A database of IN sequences may becreated and used as described in WO 01/79540. For example, such adatabase may be analysed in combination with pol and pro sequenceinformation and the results used in the determination of appropriatetreatment strategies. Said database containing a collection ofgenotypes, phenotypes and samples for which the combinedgenotype/phenotype are available may be used to determine the virtualphenotype (see supra). In addition, instead of interrogating thecomplete IN sequences, particular codons correlating to a change in drugsusceptibility of the virus may be interrogated in such database.

A primer may be chosen from SEQ ID N^(o) 1-23. A particular set ofprimers is SEQ ID 1-10, 13, 15, and 23. Primers specific for the INregion of the HIV genome such as the primers described herein and theirhomologs are claimed. The primer sequences listed herein may belabelled. Suitably, this label may be detected using fluorescence,luminescence or absorbance. The primer for creating a deletion constructmay contain a portion that does not anneal to the HIV sequence. Thatportion may be used to introduce a unique restriction site.Interestingly, primers may be designed in which the unique restrictionsite is partially present in the HIV sequence. The primers are chosenfrom those listed herein or have at least 80% homology as determined bymethods known by the person skilled in the art such BLAST or FASTA.Specifically, the homology is at least 90%, more specifically, at least95%. In addition, primers located in a region of 50 nucleotides (nt)upstream or downstream from the sequences given herein constitute partof the invention. Especially, said region is 20 nucleotides up ordownstream from the position in the HIV genome of the primer sequencesgiven herein. Alternatively, primers comprising at least 8 consecutivebases present in either of the primers described here constitute oneembodiment of the invention. Interestingly, the primers comprise atleast 12 consecutive bases present in either of the primers describedherein.

The present invention comprises the plasmids described in theexperimental part and the use of the plasmids in the methods describedherein. The HIV sequence incorporated in the plasmid may be based on theK03455 sequence. The complete HIV sequence may be incorporated or onlypart thereof. A suitable plasmid backbone may be selected from the groupincluding pUC, pSV or pGEM.

A plasmid comprising a deleted integrase, wherein the deletion comprisesat least 100 bp of the HIV integrase gene is provided herein. Suitably,more that 500 bp of the integrase gene are deleted, more suitably thecomplete IN gene is deleted. The deletion may also comprise parts offlanking genes, or eventually more than one gene, e.g. deletion ofintegrase and protease.

To prepare vectors containing recombinant IN coding sequences, thepatient derived IN RNA can be reverse transcribed and amplified by thepolymerase chain reaction (PCR), then inserted into a vector containingthe wild type HIV genome sequence but lacking a complete IN codingregion. Initially 36 different primer combinations were used to obtainamplified DNA sequences from 16 patient samples. The 5′ to 3′ sequencesand the primers identified by SEQ ID's NO 1-10 of primers that can besuccessfully used to reverse transcribe and PCR amplify IN codingregions are listed below in Table 1.

A number of reverse transcription and PCR protocols known in the art maybe used in the context of the present invention. A nested PCR approachto amplify the patient derived cDNA after reverse transcription may beused as described in Kellam, P. and Larder, B. A. , (AntimicrobialAgents and Chemotherapy 38: 23-30 (1994)), which is incorporated hereinby reference. The nested approach of the instant invention utilizes twosets of primers, the outer primers are 5′EGINT1 (SEQ ID NO 1) and3′EGINT 10 (SEQ ID NO 11), while the inner primers are 5′EGINT107 (SEQID NO 2) and 3′EGINT11 (SEQ ID NO 12). An additional inner 5′ primer,5′EGINT2 (SEQ ID NO 3), may also be used as a “rescue primer” to improvethe yield of amplified DNA. Amplification using these primers yields aPCR product encompassing the complete IN coding sequence. Alternatively,5′EGINT3 (SEQ ID NO 4) and 3′EGINT10 (SEQ ID NO 11) are used as outerPCR primers, while 5′EGINT4 (SEQ ID NO 5) or 5′EGINT5 (SEQ ID NO 6) and3′EGINT6 (SEQ ID NO 7) are used as inner primers, yielding a PCR productencompassing a portion of the IN coding sequence.

TABLE 1 Primers for IN reverse transcription and PCR amplification.Primer Name 5′ to 3′ sequence SEQ ID NO R-IN-vif and IN outer and innerprimers 5′EGINT1 GGTACCAGTTAGAGAAAGAACCCA SEQ ID NO:1 5′EGINT107GGAGCAGAAACCTTCTATGTAGATG SEQ ID NO:2 5′EGINT2 GGCAGCTAACAGGGAGACTAA SEQID NO:3 5′EGINT3 GGAATCATTCAAGCACAACCAGA SEQ ID NO:4 5′EGINT4TCTGGCATGGGTACCAGCACA SEQ ID NO:5 5′EGINT5 AGGAATTGGAGGAAATGAACAAGTA SEQID NO:6 3′EGINT6 GTTCTAATCCTCATCCTGTCT SEQ ID NO:7 3′EGINT7CCTCCATTCTATGGAGTGTCTATA SEQ ID NO:8 3′EGINT8 GGGTCTACTTGTGTGCTATATCTCSEQ ID NO:9 3′EGINT9 CAGATGAATTAGTTGGTCTGCTA SEQ ID NO:10 3′EGINT10 CCTCCA TTC TAT GGA GAC TCC CTG SEQ ID NO:11 3′EGINT11 GCA TCC CCT AGT GGGATG TG SEQ ID NO:12 R-IN-vif deletion-mutagenesis primers MUT-IN1 GGGTGA CAA CTT TTT GTC TTC CTC SEQ ID NO:13 TAT MUT-IN2 GGA TCC TGC AGC CCGGGA AAG CTA SEQ ID NO:14 GGG GAT GGT TTT ATA IN deletion-mutagenesisprimers: MUT-IN3 GGG CCT TAT CTA TTC CAT CTA AAA SEQ ID NO:15 ATA GTMUT-IN4 GGA TCC TGC AGC CCG GGA TTA TGG SEQ ID NO:16 AAA ACA GAT GGC ASequencing primers IN_SEQ1F AGT CAG TGC TGG AAT CAG G SEQ ID NO:17IN_SEQ2F TTC CAG CAG AAA CAG GGC AG SEQ ID NO:18 IN_SEQ3F GTA GAC ATAATA GCA ACA GAC SEQ ID NO:19 IN_SEQ1R CCC TGA AAC ATA CAT ATG GTG SEQ IDNO:20 IN_SEQ2R CTG CCA TTT GTA CTG CTG TC SEQ ID NO:21 IN_SEQ1R TGA ACTGCT ACC AGG ATA AC SEQ ID NO:22 The underlined portions do not anneal tothe sequence to be amplified.

To prepare recombinant vectors comprising the amplified patient-derivedIN sequences, these sequences can be inserted into vectors comprisingthe wild-type HIV sequence and a deletion of all or part of the INcoding region. The wild type HIV sequence can be obtained from a plasmidsuch as pSV40HXB2D that is capable of transfecting lymphocyte cells toproduce viable virus particles. A deletion of the entire IN codingregion on the pSV40HXB2D vector may effectively be created by PCRamplifying the plasmid using primers annealing to sequences at or nearthe ends of the IN coding region in the vector. The amplified productcan be cleaved with a restriction enzyme introduced into the primers,then re-ligated to create a pSV40HXB2D-based IN deletion vector with aunique restriction site at the location of the deletion. The IN deletionvector can have a deletion of the complete IN coding sequence,optionally with part of the preceding RNase and/or subsequent Vif codingsequences also deleted. Alternatively, a partial deletion of the INcoding sequence is created. This restriction site is unique for thecomplete plasmid including the HIV gene. An example of such restrictionsite is the SmaI restriction site. Interestingly, the primers forcreating a deletion construct are selected from SEQ ID N^(o) 13-16.

Those of skill in the art will appreciate that several types of HIVvectors and cloning procedures known in the art may be used to create INdeletion plasmids for recombination or ligation with patient derivedsequences and creation of infectious viruses. Generally, such vectorsmust be created to allow re-insertion of the deleted sequences withoutdisrupting the reading frame of the gag-pol gene.

The amplified IN sequences may be inserted into the appropriate vectorby homologous recombination between overlapping DNA segments in thevector and amplified sequence. Alternatively, the amplified IN sequencecan be incorporated into the vector at a unique restriction siteaccording to cloning procedures standard in the art. This latter is adirect cloning strategy.

EXPERIMENTAL PART Example 1 Phenotyping HIV Integrase

1. PCR Amplification of Integrase Encoding Sequence

The integrase encoding sequence was amplified from either wildtype HIV-1(IIIB) or NL4.3 virus, or HXB2D site-directed mutant viruses containingmutations in integrase (such as T66I, S153Y, M154I, or combinationsthereof) (Hazuda et al., Science 2000, 287, 646-650), or patientsamples. Starting from RNA, extracted from virus supernatant or plasmausing the QIAamp® viral RNA extraction kit (Qiagen), cDNA wassynthesized by reverse transcription (Expand™ reverse transcriptase, 30min at 42° C.) with the primer 3′EGINT10 (SEQ ID NO 11), followed by anested PCR. The outer PCR was performed with the primers 5′EGINT1 (SEQID NO 1) and 3′EGINT10 (SEQ ID NO 11) (R-IN-vif construct) or 5′EGINT3(SEQ ID NO 4) and 3′EGINT10 (SEQ ID NO 11) (IN construct) (Expand™ HighFidelity PCR system), and 5 μl of the outer product was used for aninner PCR with primers 5′EGINT2 (SEQ ID NO 3) and 3′EGINT11 (SEQ ID NO12) (R-IN-vif construct) or 5′EGINT4 (SEQ ID NO 5) and 3′EGINT6 (SEQ IDNO 7) (IN construct). In a second protocol the outer primers wereidentical as described above, the inner primers are 5′EGINT5 (SEQ ID NO6) and 3′EGINT6 (SEQ ID NO 7). The amplicons can be used for genotypingand phenotyping. Cycling conditions for both PCRs are denaturation for 3min at 95° C., followed by 30 cycles of 1 min 90° C., 30 sec 55° C., and2 min 72° C. A final extension was performed at 72° C. for 10 min. Forrecombination, PCR products are purified using the QiaQuick® 96 PCRBioRobot kit (Qiagen), according to the manufacturer's protocol. If theprotocol starts from DNA containing the HIV material such as proviralDNA, the reverse transcriptase step is not needed. The nested approachis also not needed when starting from proviral DNA. The obtainedamplicons were sequenced using the primers: In_seq1F (SEQ ID NO 17),In_seq2F (SEQ ID NO 18), In_seq3F (SEQ ID NO 19), IN_seq1R (SEQ ID NO20), IN_seq2R (SEQ ID NO 21), and IN_seq3R (SEQ ID NO 22). The sequenceof the IIIB and patient amplicon, and the NL4.3 amplicon were identicalto the reference IIIB and NL4.3 sequences respectively (data not shown).

2. Preparation of a IN Deletion Construct

A R-IN-vif or IN deletion construct was generated by site-directedmutagenesis on the template pSV40HXB2D with the primers MUT-IN1 (SEQ IDNO 13) and MUT-IN2 (R-IN-vif construct) (SEQ ID NO 14) or MUT-IN3 (SEQID NO 15) and MUT-IN4 (SEQ ID NO 16) (IN construct) (protocolSite-directed mutagenesis kit, Stratagene). After DpnI digestion forremoval of the methylated template DNA, the construct was digested withSmaI and ligated to circulize the plasmid. The plasmid was transformedinto competent cells such as Top 10 cells, and colonies were screenedfor the presence of the deletion construct. The IN-deletion constructwas checked by sequence analysis with primers 5′EGINT1 (SEQ ID NO 1) or5′EGINT10 (SEQ ID NO 11) and 3′EGINT10 (SEQ ID NO 11) or 3′EGINT11 (SEQID NO 12). For use in recombination experiments, large-scale plasmid DNApreparations were linearized by SmaI digestion and recombined with PCRamplified integrase genes from wild type, mutant, or patient viruses.The plasmid containing the integrase deletion (IN) has been depositedpSV40HXB2D-IN. The sequence of said plasmid is 14377 nucleotides long.The R-IN-vif deletion construct is 13975 nucleotides long. ThepSV40HXB2D-IN was deposited at the Belgian Coordinated Collections ofMicro-Organisms located at the Universiteit Gent—Laboratorium voorMoleculaire Biologie on Aug. 5, 2002 and the accession number is LMBP4574.

3. Recombination of Interase-amplified Sequences with the CorrespondingDeletion Construct

Recombinant virus was produced by co-transfection by electroporation ofthe SmaI-linearized IN-deletion construct and the integrase ampliconinto MT4 cells or MT4 cells equipped with an LTR driven reporter geneconstruct. Production of recombinant virus was evaluated by scoring thecytopathogenic effect (CPE) that is induced by HIV-infection of MT4cells or by the LTR-driven reporter signal induced by HIV infection inMT4 reporter cells. Green fluorescent protein was used as the reportersignal. Viruses are harvested and titrated at maximum CPE.

For recombination the deletion construct pSV40HXB2D-IN was used.Recombination experiments were performed with amplicon from wildtype HIVIIIB and NL4.3, and patient sample 146514 generated by both primer sets.For each recombination 2 μg amplicon was co-transfected with 10 μgSmaI-digested pSV40HXB2D- IN by electroportion into MT4-LTR-EGFP cells.Virus stocks were titrated and tested in an antiviral experiment on areference panel including nucleoside reverse transcriptase inhibitors(NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), proteininhibitor (PR), entry and integrase (IN) inhibitors (Table 2).

Recombination was checked by nucleic acid sequence analysis usingprotocols known to the person skilled in the art. Sequencing primerswhich can be used are In_seq1F (SEQ ID NO 17), In_seq2F (SEQ ID NO 18),In_seq3F (SEQ ID NO 19), IN_seq1R (SEQ ID NO 20), IN _seq2R (SEQ ID NO21), and IN_seq3R (SEQ ID NO 22). The recombinant virus was evaluated inan anti-viral assay with a panel of reference compounds includingnucleoside RT inhibitors (NRTI) Zidovudine (AZT) Lamivudine (3TC),Didanosine (DDI), non-nucleoside RT inhibitors (NNRTI) Nevirapine (NVP),4-[[6-amino-5-bromo-2-[(4-cyanophenyl)amino]-4-pyrimidinyl]oxy]-3,5-dimethyl-benzonitrilealso referred to as compound1,4-[[6-amino-5-bromo-2-[(4-cyanophenyl)amino]-4-pyrimidinyl]oxy]-3,5-dimethyl-Benzonitrile,also referred to as compound 2 protease inhibitors (PR) Saquinavir(SQV), Amprenavir (APV), Indinavir (IDV),[(1S,2R)-3-[[(4-aminophenyl)sulfonyl](2-methylpropyl)amino]-2-hydroxy-1-(phenylmethyl)-propyl]-, (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3yl ester carbamicacid also referred to as compound 3, entry-inhibitors (Entry)(AMD3100,DS5000, ATA), and integrase inhibitor (IN) 2-(1-methylethyloxy)- ,-dioxo-5-(phenylmethyl)-benzenebutanoic acid also referred to ascompound 4. The results are compiled in Table 2. AVE means antiviralexperiment. Type means the type of inhibitor investigated. The foldchange is the fold change in EC₅₀. WT III B means that a portion of thewild type IIIB strain has been amplified and used in the antiviralexperiment, including transfection and generation of recombinant virus.NL 4-3 means the integrase gene of this laboratory strain has beenamplified and subsequently used for the antiviral experiment. Patient146514 means that the integrase gene of an HIV sample retrieved fromsaid patient has been amplified and used in the antiviral experiment.pHXB2D has been used as a control. No recombination has been effectedusing this HIV clone. pHXB2D has been used directly for transfection andantiviral experiment. Primer set 3 consist of outer primers 5′EGINT3(SEQ ID NO 4) and 3′EGINT10 (SEQ ID NO 11), and inner primers 5′EGINT4(SEQ ID NO 5) and 3′EGINT6 (SEQ ID NO 7). Primer set 4 consist of outerprimers 5′EGINT3 (SEQ ID NO 4) and 3′EGINT10 (SEQ ID NO 11), and innerprimers 5′EGINT5 (SEQ ID NO 5) and 3′EGINT6 (SEQ ID NO 7). Othersuitable integrase inhibitors include L-731,988, diketo-acids andS-1360.

The antiviral activity of these compounds against recombinant virus fromwildtype HIV-1 IIIB or NL4.3 was identical to the activity against theHIV-1 IIIB and pHXB2D control strain, where no recombination has beenperformed. Recombinant virus generated from site-directed mutant virusgave a fold increase in EC₅₀ against compound 4 of respectively 2-fold(T66I mutation), 5-fold (S153Y), 3-fold (M154I mutation), 10-fold (T66I/S153Y mutations or T66I/ M154I mutations). Recombinant virus generatedfrom patient samples without mutations in the integrase coding sequence,displyed analogous results as the wildtype strains in the antiviralassay. The panel of protease and reverse transcriptase inhibitors wereincluded in the list to prove that no background resistance, expressedas a fold increase in EC₅₀, was detected. The reverse transcriptase andprotease genes present in the antiviral experiments were derived fromwild type HIV sequence, which does not confer resistance to the drugsincluded. The instant results (Table 2) indicate that no change insusceptibility for any of these compounds is found.

TABLE 2 Antiviral experiment Example 2 Genotyping of integrase Themethods and conditions used for sequence analysis of HIV integrase geneare outlined below. The sequencing primers (cfr. Table 1) cover theregion of 864 nucleotides, from nucleotide 4230 until 5093 according tothe sequence present in the HIV clone HXB2D. The sequencing primers werediluted until 1 pmol/μl and used in the mix and conditions as describedbelow. Reaction Mix Component Reaction Mix Big Dye Terminator Mix   4 μl2.5 Dilution Buffer   4 μl Water 4.8 μl Primer (1 pmol/μl) 3.2 μl Sample(200-500 ng/μl)   4 μl TOTAL  20 μl Thermal Cycle Conditions InitialDenaturation 3′ on 96° C. Denaturation 30″ on 96° C. Annealing 15″ on50° C. 30 cycles Elongation 4′ on 60° C. Hold on 4° C.

After cycle sequencing the reaction products were purified and run onthe 3700 DNA analyzer.

Example 3 Construction of a Recombinant IN Vector

A) Construction of pSV40HXB2D R-IN-vif

The pSV40HXB2D R-IN-vif vector has a deletion of the complete IN codingsequence as well as part of the preceding RNase and subsequent Vifcoding sequences. It was constructed by PCR amplification of pSV40HXB2Dand religation of the amplified fragment. The primers used foramplification were MUT IN1 (5′ GGG TGA CAA CTT TTT GTC TTC CTC TAT 3′;SEQ ID NO:13) and IN2 (5′ GGA TCC TGC AGC CCG GGA AAG CTA GGG GAT GGTTTT ATA GA 3′; SEQ ID NO:23), which contain a SmaI site. Primer MUT IN1(SEQ ID NO 13) anneals to nucleotides 3954 to 3928, and primer IN2 (SEQID NO 23) anneals to nucleotides 5137 to 5163. The first 14 nucleotidesof IN2 (SEQ ID NO 23) comprise the Sma I tail, which does not anneal tothe vector. The amplified product was cleaved with Sma I and re-ligatedto create pSV40HXB2D R-IN-vif, with a Sma I recognition site at thelocation of the deletion.

B) Amplification of Patient Derived IN Sequences for Insertion intopSV40HXB2D R-IN-vif

To amplify the complete IN coding region and the flanking segments ofthe RNase and Vif coding regions for insertion into the pSV40HXB2DR-IN-vif vector, a nested PCR method was used. The outer primers were5′EGINT1 (SEQ ID NO 1) and 3′EGINT10 (SEQ ID NO 11), while the inner setwas 5′EGINT107 (SEQ ID NO 2) and 3′EGINT11 (SEQ ID NO 12). An additionalinner 5′ primer, 5′EGINT2(SEQ ID NO 3), was used to improve the yield ofamplified DNA. (The sequences of these primers are given in Table 1,above.)

C) Construction of the pSV40HXB2D IN Vector

To create pSV40HXB2D IN, the pSV40HXB2D vector was PCR amplified andre-ligated to effectively delete most of the IN coding region, leavingthe nucleotides coding for the N-terminal 8 amino acids and theC-terminal 20 amino acids in place. The amplification was performedusing the primers MUT IN3 (5′ GGG CCT TAT CTA TTC CAT CTA AAA ATA GT 3′;SEQ ID NO:15) and MUT IN4 (5′GGA TCC TGC AGC CCG GGA TTA TGG AAA ACA GATGGC A 3′; SEQ ID NO:16), containing a SmaI site. Primer MUT IN3 (SEQ IDNO 15) anneals to nucleotides 4254 to 4226, and primer MUT IN4 (SEQ IDNO 16) anneals to nucleotides 5 create 036 to 5057. The resultingamplified fragment can be cleaved with SmaI and re-ligated to pSV40HXB2DIN.

D) Amplification of Patient Derived IN Sequences for Insertion intopSV40HXB2D IN

Patient derived IN sequences was prepared for insertion into the HIVdeletion vector using a nested PCR approach as in part B above. 5′EGINT3(SEQ ID NO 4) and 3′EGINT10 (SEQ ID NO 11) were used as outer PCRprimers, while 5′EGINT4 (SEQ ID NO 5) or 5′3GINT5 (SEQ ID NO 6) and3′EGINT6 (SEQ ID NO 7) were used as inner primers. The sequences and SEQID NO 4-8 of these primers are given in Table 1. The underlined portionof MUT IN4 (SEQ ID NO 16) represents the SmaI tail that does not annealto the vector.

E) Homologous Recombination and Ligation to Insert the PCR Products intothe Vectors

The pSV40HXB2D IN or pSV40HXB2DAR-IN-vif vectors was linearized withSmaI. The vectors and the amplified IN DNA fragments were transfected byelectroporation into MT4 cells, MT4 cells equipped with a LTR reportergene construct (MT4-rep) or PM-1 cells. By homologous recombinationbetween overlapping portions of the vector and IN amplicons, the HIVgenome was reconstituted with a patient derived IN coding region. Therecombinant vectors were capable of producing virus particles ininfected cells. Virus production was evaluated by scoring thecytopathogenic effect (CPE) that was normally induced by HIV infectionof MT4, MT4-rep, or PM-1 cells, or was evaluated by the inducedLTR-driven reporter signal in MT4-rep or PM-1 cells. Homologousrecombination with wild type IN sequences was used as a control.

The presence of recombinant IN DNA and RNA sequences in the transfectedcells was monitored by reverse transcription and PCR analysis. Thepresence of PCR products corresponding to correctly inserted INsequences showed that recombination successfully occurred and that viralRNA was produced in the cells.

Patient derived IN sequences and wild type controls were alternativelyinserted into SmaI-linearized pSV40HXB2D N or pSV40HXB2D R-IN-vifvectors by a standard restriction digestion and ligation procedure. TheIN amplicons were modified to create SmaI cleaved ends and were theninserted by ligation into the SmaI site on the vectors.

Example 4 Genotyping of Patient Derived IN Coding Sequences

A) Obtaining and Amplifying Patient Derived IN Sequences

RNA was isolated from 100 μl of plasma according to the method describedby Boom et al. (1990), and reverse transcribed with the GENEAMP® reversetranscriptase kit (Perkin Elmer) as described by the manufacturer usingan HIV-1 specific downstream primer. Two subsequent nested PCRs were setup using specific outer primers and inner primers, respectively. Theouter primer reaction were performed as described in WO97/27480 and U.S.Pat. No. 6,221,578 (which are incorporated herein by reference). Theinner amplification was performed in a 96 well plate as follows: 4 μl ofthe outer amplification product was diluted to a final volume of 50 μlusing a 10× amplification mix consisting of 5 μl 10×PCR buffercontaining 15 mM MgCl₂, 1 μl dNTP's (10 mM) 0.5 μl each primer (0.25μg/ml), 0.4 μl EXPAND® High Fidelity polymerase (3.5 U/μl; Roche) anddeionized water. Amplification was initiated after a short denaturationof the amplification product made using the outer primers (2 min at 94°C). Ten amplification cycles were run, each consisting of a 15 secdenaturation step at 94° C., a 30 sec annealing step at 60° C. and a 2min polymerization step at 72° C. This amplification was immediatelyfollowed by 25 cycles consisting of a 15 sec denaturation step at 94°C., a 30 sec annealing step at 60° C. and a variable time polymerizationstep at 72° C. The polymerization step was initially run for 2 min and 5sec, then was increased by 5 seconds in each cycle. Amplification wascompleted by an additional polymerization step of 7 min at 72° C. Thereactions were held at 4° C. until further analysis or stored at −20° C.(for short periods) or −70° C. (for longer periods). The products can beanalysed on DNA agarose gels and visualised by UV-detection. Theproducts can be purified using the QIAQUICK® 96-well plate system asdescribed by the manufacturer (Qiagen).

B. Sequencing of IN Coding Region

The IN coding region present on the amplified fragments were sequencedusing techniques known in the art. The sequencing was started by firstdistributing 4 μl of the primer stocks (4.0 μM) over a 96 well platewhere each stock was pipetted down the column. In a second step, mastermixes were made consisting of 14 μl deionized water, 17.5 μl dilutionbuffer, 7 μl sample (PCR fragment) and 14 μl Big Dye™ Terminator Mix(Perkin Elmer). A fraction (7.5 μl) of each master mix, containing aspecific PCR fragment, was transferred to a specific place into the 96well plate so that each sample fraction was mixed with a different PCRprimer set. Samples were pipetted across the rows. Samples were placedin a thermal cycler and sequencing cycles started. The sequencingreaction consisted of 25 repetitive cycles of 10 sec at 96° C., 5 sec at50° C. and 4 min at 60° C., respectively. Finally, sequence reactionswere be held at 4° C. or frozen until further analysis. The sequencingreactions were precipitated using a standard ethanol precipitationprocedure, resuspended in 2 μl formamide and heated for 2 minutes at 92°C. in the thermal cycler. Samples were cooled on ice until ready toload. 1 μl of each reaction was loaded on a 4.25% vertical acrylamidegel in a 377 sequencer system and gel was run until separation of thefragments is complete.

C. Sequence Analysis of IN Coding Region

Sample sequences wer imported as a specific project into the sequencemanager of Sequencher™ (Genecodes) and compared to the wild typereference sequence. Sequences were assembled automatically and set at85% minimum match. Secondary peaks were searched and the minimum was setat 60%. Any sequence that extended beyond the 5′ end or the 3′ end ofthe reference were deleted. When a region of overlap between sequencesfrom the same strand was reached, the poorest quality of sequence wasdeleted leaving an overlap of 5-10 bases. Ambiguous base calls wereconsidered poor matches to exact base calls. The sequence assembly wassaved within an editable contig.

Obtained sequences were edited to facilitate interpretation of the basecalls. Ambiguous sequences were retrieved and checked for possibleerrors or points of heterogeneity. When the point of ambiguity appearedcorrect (both strands of sequence agreed but were different from thereference sequence) it was interpreted to be a variant. The referencesequence was used as an aid for building a contig and as a guide tooverall size and for trimming. The reference sequence was not used fordeciding base calls. A change was only made when both strands agreed.All gaps were deleted or filled, unless they occurred in contiguousgroups of multiples of three (i.e., insertion or deletion of completecodons) based on data form both sequence strands. Once the editing wascomplete, the new contig sequence was saved as a consensus sequence andused for further analysis.

Detailed sequence editing was performed following certain rules: A)Applied Biosystems, Inc. primer blobs were trimmed at 5′ ends where 1consecutive base remained off the scale, the sequence was trimmed notmore than 25% until the first 25 bases contained less than 1 ambiguity,at least the first 10 bases from the 5′ end were removed, and B) 3′ endswere trimmed starting 300 bases after the 5′ trim, the first 25 basescontaining more than 2 ambiguities were removed, the 3′ end was trimmeduntil the last 25 bases contained less than 1 ambiguity. The maximumlength of the obtained sequence fragment after trimming was 550 bases.

Sequences that failed to align were removed from the assembly andreplaced by data retrieved from new sequence analyses. When furtherfailures occur, PCR reactions were repeated. Chromatograms werevisualised using an IBM software system.

Legends to the Figures

FIG. 1: Overview of the HIV genome indicating the primer positions

1. A method for determining the susceptibility of at least one HIV to atleast one drug, comprising: i) obtaining at least one sample comprisingHIV RNA from a patient, wherein the sample comprises at least one INgene or a portion thereof; ii) reverse-transcribing and amplifying saidHIV RNA with primers specific for the IN region of the HIV genome toobtain an amplicon comprising the IN gene or a portion thereof, whereinat least one primer is selected from SEQ ID NO: 1-12 and 17-22; iii)determining the nucleotide sequence of the amplicon or a portionthereof, and iv) comparing the nucleotide sequence of the amplicon tothe sequence of known HIV sequences to estimate the susceptibility of atleast one HIV to as least one drug.
 2. A method for determining thesusceptibility of at least one HIV to at least one drug, comprising: i)obtaining at least one sample comprising HIV DNA from a patient, whereinthe sample comprise at least one IN gene or a portion thereof, whereinat least one primer is selected from SEQ ID NO: 1-12 and 17-22; ii)amplifying said HIV DNA with primers specific for the IN region of theHIV genome to obtain an amplicon comprising the IN gene or a portionthereof; iii) determining the nucleotide sequence of the amplicon or aportion thereof, and iv) comparing the nucleotide sequence of theamplicon to the sequence of know HIV sequences to estimate thesusceptibility of at least one HIV to at least one drug.
 3. A kit usefulfor determining the susceptibility of at least one HIV to at least onedrug according to any claim 1 or 2, comprising at least one primerselected from SEQ ID NO: 1-12 and 17-22.
 4. A primer as selected fromSEQ ID NO 1-14.
 5. A primer selected from SEQ ID NO 1-23.
 6. A primerselected from SEQ ID NO 1-7 and SEQ ID NO 11-22.